The overall goal of this study is the elucidation of the molecular interactions and events underlying the cascade of herpes simplex virus (HSV-1) transcription during productive infection. Despite knowing of the existence of this regulatory cascade for 4 decades, our knowledge of the molecular underpinning for it is strikingly lacking. Detailed knowledge of how HSV genes are transcribed, and the virus-cell interactions that contribute to their regulated expression, may allow for strategies to block activated transcription and hence virus multiplication. The viral genome is the only component of the virus present throughout its productive life cycle. However, the viral and cellular proteins that bind to, and th processes that occur on the genome change as the life cycle progresses from entry to the packaging of viral genomes into nascent virions. We have developed an approach, which is a modification of a technique called isolation of proteins on nascent DNA (iPOND) to determine in a nonbiased way the viral and cellular proteins that associate with the genome throughout infection, from the initial sensing of the genome to its packaging. Of relevance to this proposal are the viral and cellular proteins involved in RNA polymerase II transcription that associate with the genome, as infection proceeds. Thus, the first aim is to interrogate the viral and cellular proteins that bind to the genome throughout infection and differentiate between those binding at replication forks and those binding to replicated DNA. The viral Infected Cell Polypeptide 4 (ICP4) is large multifunctional transcription regulator that binds to the viral genome. The cellula proteins/complexes that affinity-purify with ICP4 change as infection proceeds, and are involved in all stages of transcription. The most abundant of these are the most fundamental of general Pol II transcription factors, TFIID and mediator. Mediator(s) are variable multicomponent complexes that can positively and negatively affect the rates of transcription initiation and elongation. ICP4 may interact with multiple components/forms of mediator to differentially regulate viral genes. The second aim determines the forms of mediator that interact with ICP4 and the genome and to test the hypothesis these interactions contribute to activation and turn off of different HSV gene in the regulatory cascade. Multiple chromatin remodeling complexes were found on the viral genomes, some of which interact with ICP4. Histones appeared to be excluded, while ICP4 is one of the most abundant proteins on the genome. Preliminary ChIP-Seq experiments show that as DNA replication proceeds, the density of ICP4 binding is reduced and Pol II occupation of late genes is increased. The third aim addresses the hypothesis that ICP4 is both a mediator of cellular chromatin, helping to keep transcribing and replicating DNA free of histones, and that it also serves as a type of viral chromatin to regulate the expression o late genes. Together these studies will address the understudied more dynamic aspects of the regulatory cascade of HSV transcription.